Many medically significant biological processes that are mediated by proteins participating in signal transduction pathways involving G-proteins and/or second messengers, e.g., cAMP, have been established (Lefkowitz, 1991, Nature, 351:353-354). These proteins are often referred to as proteins participating in pathways with G-proteins or PPG proteins. Some examples of these proteins include the G protein-coupled receptors (GPCR), such as those for adrenergic agents and dopamine (B. K. Kobilka et al., 1987, Proc. Natl. Acad. Sci. USA, 84:46-50; B. K. Kobilka et al., 1987, Science, 238:650-656; and J. R. Bunzow et al., 1988, Nature, 336:783-787), G-proteins themselves, effector proteins, e.g., phospholipase C, adenylate cyclase and phosphodiesterase and actuator proteins, e.g., protein kinase A and protein kinase C (M. I. Simon et al., 1991, Science, 252:802-8).
For example, in one form of signal transduction, the effect of hormone binding results in activation of the enzyme adenylate cyclase inside the cell. Enzyme activation by hormones is dependent on the presence of the nucleotide GTP, where GTP also influences hormone binding. A G-protein binds the hormone receptors to adenylate cyclase. The G-protein has further been shown to exchange GTP for bound GDP when activated by hormone receptors. The GTP-carrying form of the G-protein then binds to an activated adenylate cyclase. Hydrolysis of GTP to GDP, catalyzed by the G-protein itself, returns the G-protein to its basal, inactive form. Thus, the G-protein serves a dual role—as an intermediate that relays the signal from receptor to effector, and as a “clock” that controls the duration of the signal.
The membrane protein gene superfamily of G-protein coupled receptors (GPCRs) has been characterized as having seven putative transmembrane domains. The domains are believed to represent transmembrane α-helices connected by extracellular or cytoplasmic loops. GPCRs include a wide range of biologically active receptors, such as hormone, viral, growth factor, and neuronal receptors.
GPCRs are further characterized as having seven conserved hydrophobic stretches of about 20 to 30 amino acids, connecting at least eight divergent hydrophilic loops. The G-protein coupled receptor family includes, for example, the following types of receptors: dopamine, calcitonin, adrenergic, endothelin, cAMP, adenosine, muscarinic, acetylcholine, serotonin, histamine, thrombin, kinin, follicle stimulating hormone, opsins, endothelial differentiation gene-1 receptor, rhodopsins, odorant and cytomegalovirus receptors, etc.
Most GPCRs have single conserved cysteine residues in each of the first two extracellular loops which form disulfide bonds that are believed to stabilize functional protein structure. The 7 transmembrane regions are designated as TM1, TM2, TM3, TM4, TM5, TM6, and TM7. TM3 has been implicated in signal transduction. Phosphorylation and lipidation (palmitylation or farnesylation) of cysteine residues can influence signal transduction of some GPCRs. Most GPCRs contain potential phosphorylation sites within the third cytoplasmic loop and/or the carboxyl terminus. For several GPCRs, such as the β-adrenoreceptor, phosphorylation by protein kinase A and/or specific receptor kinases mediates receptor desensitization.
For some receptors, the ligand binding sites of GPCRs are believed to comprise a hydrophilic socket formed by the transmembrane domains of several GPCRs. This socket is surrounded by hydrophobic residues of the GPCRs. The hydrophilic side of each GPCR transmembrane helix is postulated to face inward and form the polar ligand-binding site. TM3 has been implicated in several GPCRs as having a ligand-binding site, which includes the TM3 aspartate residue. In addition, serines within TM5, a TM6 asparagine and phenylalanines or tyrosines within TM6 or TM7 are also implicated in ligand binding.
GPCRs can be intracellularly coupled by heterotrimeric G-proteins to various intracellular enzymes, signal transduction pathways and molecules, ion channels and transporters (see, e.g., Johnson et al., 1989, Endocrin. Rev., 10:317-331). Different G-protein β-subunits preferentially stimulate particular effectors to modulate various biological functions in a cell. Phosphorylation of cytoplasmic residues of GPCRs have been identified as an important mechanism for the regulation of G-protein coupling of some GPCRs. GPCRs are found in numerous sites within a mammalian host.
GPCRs are one of the largest receptor superfamilies known. These receptors are biologically important and malfunction of these receptors results in diseases such as Alzheimer's, Parkinson, diabetes, dwarfism, color blindness, retinitis pigmentosa, asthma and others. GPCRs are also involved in depression, schizophrenia, sleeplessness, hypertension, anxiety, stress, renal failure and in several other cardiovascular, metabolic, neural, oncological and immune disorders (F. Horn and G. Vriend, 1998, J. Mol. Med., 76:464-468). They have also been shown to play a role in HIV infection (Y. Feng et al., 1996, Science, 272: 872-877).
As mentioned above, the structure of GPCRs comprises seven transmembrane helices that are connected by loops. The N-terminus is always extracellular and C-terminus is intracellular. GPCRs are involved in signal transduction. The signal is typically received at the extracellular N-terminus side. The signal can be an endogenous ligand, a chemical moiety, another type of extracellular signal, and even light. This signal is then transduced through the membrane to the cytosolic side where a heterotrimeric G-protein is activated which in turn elicits a response (F. Horn et al., 1998, Recept. and Chann., 5: 305-314).
Modulators, e.g., ligands, agonists and antagonists, for GPCRs are utilized for preventative and therapeutic purposes to treat various diseases, disorders and/or conditions. The present invention has achieved the identification of a particular GPCR, newly linked by this invention to chronic obstructive pulmonary disease (COPD) and COPD-related disorders and conditions. This COPD-related GPCR, as described herein and newly associated with COPD, provides a new target for drug discovery and treatments effective against COPD and disorders and conditions related thereto. In addition, modulators of this COPD-related GPCR have been newly found in accordance with the present invention and as described herein to affect transcriptional mediators and signaling molecules, thereby providing new agents for treating not only COPD, but also other diseases and conditions affected by regulation of these transcriptional mediators, components of pathways related thereto and/or cell signaling components.
Chronic obstructive pulmonary disease (COPD), which encompasses both chronic bronchitis and emphysema, is one of the most common respiratory conditions of adults in the developed world. COPD poses an enormous burden to society both in terms of direct cost to healthcare services and indirect costs to society, primarily through loss of productivity. In the Western world, COPD is the fourth most common cause of death and it claims the lives of over 119,000 Americans annually. Approximately eighty to ninety percent of COPD patients have smoked and/or do smoke cigarettes.
The definition of COPD that is recognized by both the American Thoracic Society and the European Respiratory Society is a disorder characterized by reduced maximal expiratory flow and slow forced emptying of the lungs—features that do not change markedly over several months. This limitation in airflow is only minimally reversible with bronchodilators. The respiratory disease emphysema is defined pathologically as a condition in which there is permanent destructive enlargement of the air spaces distal to the terminal bronchioles without obvious fibrosis. Chronic bronchitis is defined clinically by the presence of chronic bronchial secretions, enough to cause expectoration, occurring on most days for a minimum of three months of the year for two consecutive years. The pathological basis of chronic bronchitis is mucus hypersecretion secondary to hypertrophy of the glandular elements of the bronchial mucosa. Patients with COPD have features of both conditions, although one of the conditions may be more prominent than the other.
Chronic bronchitis only really became recognized as a distinct disease, rather than as a set of symptoms, in the late 1950's. The great ‘British Smogs’ of the 1950's precipitated the deaths of many patients from respiratory failure. There is little doubt that at the present time, the most important risk factor in the development of COPD is smoking of tobacco burning products, in particular, cigarette smoking. Because approximately 80 to 90% of COPD cases are caused by smoking, a smoker is ten times more likely than a nonsmoker to die of COPD. According to the World Health Organization, 75% of deaths from COPD that occur in developed countries are directly related to smoking tobacco.
The effects of smoke from tobacco burning products, such as cigarette smoke, on the lungs are manifold. Cigarette smoke has been found to attract inflammatory cells into the lungs and stimulates the release of the proteolytic enzyme elastase from these cells. Elastase, in turn, breaks down elastin, a normal structural component of lung tissue. Normally, however, the lung is protected from the destructive effect of elastase by an inhibitor, alpha-1 antitrypsin (AAT). One effect of cigarette smoke is to attract more cells and stimulate the release of more elastase. The development of COPD, and in particular emphysema, is thought to be due, in part, to the imbalance between the destructive elastase and the protective AAT.
Not all people who smoke develop COPD; and not all patients with COPD are smokers, or have smoked in the past (although 85% to 90% of COPD patients have smoked or are smoking). There seems to be a varying susceptibility to lung damage due to cigarette smoke within the general population. Only a proportion of smokers (maybe only 10-15%) or former smokers show a rate of decline of lung function over the years that is fast enough to result in the severe impairment that is typical of patients who present with breathlessness due to COPD. Unfortunately, these types of former smokers do not improve after they stop smoking. By the time these subjects are symptomatic with breathlessness, they will have already had severe impairment of lung function. Cessation of smoking at this stage may extend their life expectancy, but may not improve their symptoms.
Another well established risk factor for COPD is a deficiency of the protective protease inhibitor, alpha-1 antitrypsin (AAT), which is produced in the liver. The risk factor relates to an inherited autosomal recessive (designated PiZZ) disorder which is fairly rare in the general gene pool. The incidence of homozygous births is about 1 in 3000 live births. As such, AAT deficiency accounts for probably less than 5% of all cases of COPD. The onset of AAT deficiency emphysema generally occurs between the ages of 20 to 40 years and is characterized by shortness of breath and decreased exercise capacity. Blood screening is used if the trait is suspected and can determine if a person is a carrier, or is AAT-deficient. If children are diagnosed as being AAT-deficient through blood screening, they may undergo a liver transplant.
Low levels of AAT allow the uninhibited action of elastase on the lung parenchyma, thereby giving rise to destruction of the alveoli and the eventual development of emphysema rather than chronic bronchitis. The pattern of emphysema in AAT deficiency differs slightly from that of smoking-induced pure emphysema in that AAT deficiency produces panlobular emphysema affecting predominantly the lower lung fields, while smoking-induced emphysema is usually centrilobular affecting the upper lung fields initially.
The quality of life for a person suffering from COPD diminishes as the disease progresses. People with COPD may eventually require supplemental oxygen and may have to rely on mechanical respiratory assistance. A recent American Lung Association survey revealed that about half of all COPD patients (i.e., 51%) indicate that their condition limits their ability to work. It also limits them in normal physical exertion (70%), household chores (56%), social activities (53%), sleeping (50%), and family activities (46%).
None of the existing medications for COPD have been shown to modify the long-term decline in lung function that is the hallmark of this disease. Therefore, pharmacotherapy for COPD is used to decrease symptoms and/or complications. Bronchodilator medications are central to the symptomatic management of COPD. Additional treatment includes antibodies, oxygen therapy, and systemic glucocorticosteroids. The efficacy of inhaled glucocorticosteroids is currently under study. Chronic treatment with steroids is avoided because of an unfavorable benefit-to-risk ratio. Lung transplantation is being performed in increasing numbers and may be an option for people who suffer from severe emphysema. In addition, lung volume reduction surgery has shown promise and is being performed with increasing frequency. However, a recent study found that emphysema patients who have severe lung obstruction with either limited ability to exchange gas when breathing, or damage that is evenly distributed throughout their lungs, are at high risk of death from the foregoing procedures. Treatments for AAT deficiency emphysema, including AAT replacement therapy and gene therapy, are currently being evaluated.
Because of the magnitude of the health and health care problems that correlate with COPD and COPD related conditions and disorders, both at the level of the patient and the patient's care by the medical community, it is clear that new drug target molecules, as well as alternative drugs and treatments, are sorely needed to combat and counteract this disease. As discussed above, the present therapies are sometimes only palliative, and do not satisfactorily treat, reduce, ameliorate, or eliminate all of the debilitating effects of COPD.
In addition, as described herein, it is newly recognized that the prevalent use of tobacco burning materials and substances, such as cigarettes and cigars, generates smoke-related cellular products, e.g., proteins and peptides, which are major causative factors for COPD. Therefore, identifying the proteins, signal transduction pathways and components thereof that are activated and/or modified when cells are exposed to smoke from tobacco burning products can be key to identifying new drug targets for the treatment of COPD. The present invention newly provides previously unrecognized sources and targets for new anti-COPD drugs and compounds useful in profoundly needed treatments and therapies for COPD and COPD related diseases, which can benefit vast numbers of COPD patients and sufferers.
In their diverse cellular roles, GPCRs can also be involved in cell suicide, or programmed cell death, during the lifetime of a multicellular organism. Programmed cell death or apoptosis occurs during a number of events in the organism's life cycle, such as for example, in the development of an embryo, during the course of an immunological response, or in the demise of cancerous cells after drug treatment, among others. The final outcome of cell survival versus apoptosis is dependent on the balance of two counteracting events, namely, the onset and speed of caspase cascade activation (essentially a protease chain reaction), and the delivery of anti-apoptotic factors which block the caspase activity (B. B. Aggarwal, 2000, Biochem. Pharmacol., 60:1033-1039; N. A. Thornberry and Y. Lazebnik, 1998, Science, 281:1312-1316).
The production of anti-apoptotic proteins is controlled by the transcriptional factor complex NF-κB. For example, exposure of cells to the protein tumor necrosis factor (TNF) can signal both cell death and survival, an event playing a major role in the regulation of immunological and inflammatory responses (S. Ghosh et al., 1998, Annu. Rev. Immunol., 16:225-260; N. Silverman and T. Maniatis, 2001, Genes and Dev., 15:2321-2342; and V. Baud and M. Karin, 2001, Trends Cell Biol., 11:372-377). The anti-apoptotic activity of NF-κB is also crucial to oncogenesis and to chemo- and radio-resistance in cancer (A. S. Baldwin, 2001, J. Clin. Invest., 107:241-246).
Nuclear Factor-κB (NF-κB), is composed of dimeric complexes of p50 (NF-κB1) or p52 (NF-κB2) that are usually associated with members of the Rel family (p65, c-Rel, Rel B) which have potent transactivation domains. Different combinations of NF-κB/Rel proteins bind to distinct κB sites to regulate the transcription of different genes. Early work involving NF-κB suggested that its expression was limited to specific cell types, particularly in stimulating the transcription of genes encoding kappa immunoglobulins in B lymphocytes. However, it has been discovered that NF-κB is, in fact, present and inducible in many, if not all, cell types and that it acts as an intracellular messenger capable of playing a broad role in gene regulation as a mediator of inducible signal transduction. Specifically, it has been demonstrated that NF-κB plays a central role in the regulation of intercellular signals in many cell types. For example, NF-κB has been shown to positively regulate the human beta-interferon (beta-IFN) gene in many, if not all, cell types. Moreover, NF-κB has also been shown to serve the important function of acting as an intracellular transducer of external influences.
The transcription factor NF-κB is sequestered in an inactive form in the cytoplasm as a complex with its inhibitor, IκB; the most prominent member of the class of IκB inhibitors is IκBα. A number of factors are known to serve the role of stimulators of NF-κB activity, such as, for example, tumor necrosis factor (TNF). After TNF exposure, the inhibitor is phosphorylated and proteolytically removed, thus releasing NF-κB into the nucleus and allowing its transcriptional activity. Numerous genes are up-regulated by this transcription factor, among them IκBα. The newly synthesized IκBα protein inhibits NF-κB, effectively shutting down further transcriptional activation of its downstream effectors.
However, as mentioned above, the IκBα protein may only inhibit NF-κB in the absence of IκBα stimuli, such as TNF stimulation, for example. Other agents that are known to stimulate NF-κB release, and thus NF-κB activity, are bacterial lipopolysaccharide, extracellular polypeptides, chemical agents, such as phorbol esters, which stimulate intracellular phosphokinases, inflammatory cytokines, IL-1, oxidative and fluid mechanical stresses, and Ionizing Radiation (S. Basu et al., 1998, Biochem. Biophys. Res. Commun., 247(1):79-83). Therefore, as a general rule, the stronger the insulting stimulus, the stronger the resulting NF-κB activation, and the higher the level of IκBα transcription. As a consequence, measuring the level of IκBα RNA can be used as a marker for anti-apoptotic events, and indirectly, for the onset and strength of pro-apoptotic events.